Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity

ABSTRACT

The present invention relates to the field of glycosylation engineering of proteins. More particularly, the present invention relates to glycosylation engineering to generate proteins with improved therapeutic properties, including antibodies with increased antibody-dependent cellular cytotoxicity.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the field of glycosylationengineering of proteins. More particularly, the present inventionrelates to glycosylation engineering to generate proteins with improvedtherapeutic properties, including antibodies with increasedantibody-dependent cellular cytotoxicity.

[0003] 2. Background Art

[0004] Glycoproteins mediate many essential functions in human beings,other eukaryotic organisms, and some prokaryotes, including catalysis,signaling, cell-cell communication, and molecular recognition andassociation. They make up the majority of non-cytosolic proteins ineukaryotic organisms. (Lis et al., Eur. J. Biochem. 218:1-27 (1993)).Many glycoproteins have been exploited for therapeutic purposes, andduring the last two decades, recombinant versions ofnaturally-occurring, secreted glycoproteins have been a major product ofthe biotechnology industry. Examples include erythropoietin (EPO),therapeutic monoclonal antibodies (therapeutic mAbs), tissue plasminogenactivator (tPA), interferon-β, (IFN-β), granulocyte-macrophage colonystimulating factor (GM-CSF), and human chorionic gonadotrophin (hCG).(Cumming et al., Glycobiology 1:115-130 (1991)).

[0005] The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins et al.,Nature Biotechnol. 14:975-81 (1996)).

[0006] Mammalian cells are the preferred hosts for production oftherapeutic glycoproteins, due to their capability to glycosylateproteins in the most compatible form for human application. (Cumming etal., Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol.14:975-81 (1996)). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the bloodstream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum-free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NS0- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins et al., Nature Biotechnol.14:975-81 (1996)).

[0007] All antibodies contain carbohydrate structures at conservedpositions in the heavy chain constant regions, with each isotypepossessing a distinct array of N-linked carbohydrate structures, whichvariably affect protein assembly, secretion or functional activity.(Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). Thestructure of the attached N-linked carbohydrate varies considerably,depending on the degree of processing, and can include high-mannose,multiply-branched as well as biantennary complex oligosaccharides.(Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32 (1997)).Typically, there is heterogeneous processing of the core oligosaccharidestructures attached at a particular glycosylation site such that evenmonoclonal antibodies exist as multiple glycoforms. Likewise, it hasbeen shown that major differences in antibody glycosylation occurbetween cell lines, and even minor differences are seen for a given cellline grown under different culture conditions. (Lifely, M. R. et al.,Glycobiology 5(8):813-22 (1995)).

[0008] Unconjugated monoclonal antibodies (mAbs) can be useful medicinesfor the treatment of cancer, as demonstrated by the U.S. Food and DrugAdministration's approval of Rituximab (Rituxan™; IDEC Pharmaceuticals,San Diego, Calif., and Genentech Inc., San Francisco, Calif.), for thetreatment of CD20 positive B-cell, low-grade or follicular Non-Hodgkin'slymphoma, and Trastuzumab (Herceptin™; Genentech Inc,) for the treatmentof advanced breast cancer (Grillo-Lopez, A. -J., et al., Semin. Oncol.26:66-73 (1999); Goldenberg, M. M., Clin. Ther. 21:309-18 (1999)). Thesuccess of these products relies not only on their efficacy but also ontheir outstanding safety profiles (Grillo-Lopez, A. -J., et al., Semin.Oncol. 26:66-73 (1999); Goldenberg, M. M., Clin. Ther. 21:309-18(1999)). In spite of the achievements of these two drugs, there iscurrently a large interest in obtaining higher specific antibodyactivity than what is typically afforded by unconjugated mAb therapy.

[0009] One way to obtain large increases in potency, while maintaining asimple production process and potentially avoiding significant,undesirable side effects, is to enhance the natural, cell-mediatedeffector functions of mabs by engineering their oligosaccharidecomponent (Umaña, P. et al., Nature Biotechnol. 17:176-180 (1999)). IgGltype antibodies, the most commonly used antibodies in cancerimmunotherapy, are glycoproteins that have a conserved N-linkedglycosylation site at Asn297 in each CH2 domain. The two complexbi-antennary oligosaccharides attached to Asn297 are buried between theCH2 domains, forming extensive contacts with the polypeptide backbone,and their presence is essential for the antibody to mediate effectorfunctions such as antibody dependent cellular cytotoxicity (ADCC)(Lifely, M. R., et al., Glycobiology 5:813-822 (1995); Jefferis, R., etal., Immunol Rev. 163:59-76 (1998); Wright, A. and Morrison, S. L.,Trends Biotechnol. 15:26-32 (1997)).

[0010] The present inventors showed previously that over expression inChinese hamster ovary (CHO) cells ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umaña, P. et al., Nature Biotechnol.17:176-180 (1999), International Publication No. WO 99/54342, the entirecontents of each of which are hereby incorporated by reference in theirentirety). The antibody chCE7 belongs to a large class of unconjugatedmAbs which have high tumor affinity and specificity, but have too littlepotency to be clinically useful when produced in standard industrialcell lines lacking the GnTIII enzyme (Umana, P., et al., NatureBiotechnol. 17:176-180 (1999)). That study was the first to show thatlarge increases of maximal in vitro ADCC activity could be obtained byincreasing the proportion of constant region (Fc)-associated, bisectedoligosaccharides above the levels found in naturally occurringantibodies. To determine if this fading could be extrapolated to anunconjugated mAb, which already has significant ADCC activity in theabsence of bisected oligosaccharides, the present inventors have appliedthis technology to Rituximab, the anti-CD20, IDEC-C2B8 chimericantibody. The present inventors have likewise applied the technology tothe unconjugated anti-cancer mAb chG250.

BRIEF SUMMARY OF THE INVENTION

[0011] The present inventors have now generated new glycosylationvariants of the anti-CD20 monoclonal antibody (mAb) IDEC-C2B8(Rituximab) and the anti-cancer mAb chG250 using genetically engineeredmAb-producing cell lines that overexpressN-acetylglucosaminyltransferase III (GnTIII; EC 2.1.4.144) in atetracycline regulated fashion. GnTIII is required for the synthesis ofbisected oligosaccharides, which are found at low to intermediate levelsin naturally-occurring human antibodies but are missing in mAbs producedin standard industrial cell lines. The new glycosylated versions outperformed Mabthera™ (the version of Rixtuximab marketed in Europe) andmouse-myeloma derived chG250 in biological (ADCC) activity. For example,a ten-fold lower amount of the variant carrying the highest levels ofbisected oligosaccharides was required to reach the maximal ADCCactivity as Mabthera™. For chG250, the variant carrying the highestlevels of bisected oligosaccharides mediated significant ADCC activityat a 125-fold lower concentration than that required to detect even lowADCC activity by the unmodified control chG250. A clear correlation wasfound between the level of GnTIII expression and ADCC activity.

[0012] Accordingly, in one aspect the claimed invention is directed to ahost cell engineered to produce a polypeptide having increasedFc-mediated cellular cytotoxicity by expression of at least one nucleicacid encoding β(1,4)-N-acetylglucosaminyltransferase III (GnT III),wherein the polypeptide produced by the host cell is selected from thegroup consisting of a whole antibody molecule, an antibody fragment, anda fusion protein which includes a region equivalent to the Fc region ofan immunoglobulin, and wherein the GnT III is expressed in an amountsufficient to increase the proportion of said polypeptide carryingbisected hybrid oligosaccharides or galactosylated complexoligosaccharides or mixtures thereof in the Fc region relative topolypeptides carrying bisected complex oligosaccharides in the Fcregion.

[0013] In a preferred embodiment, the polypeptide is IgG or a fragmentthereof, most preferably, IgG1 or a fragment thereof. In a furtherpreferred embodiment, the polypeptide is a fusion protein that includesa region equivalent to the Fc region of a human IgG.

[0014] In another aspect of the claimed invention, a nucleic acidmolecule comprising at least one gene encoding GnTIII has beenintroduced into the host cell. In a preferred embodiment, at least onegene encoding GnTIII has been introduced into the host cell chromosome.

[0015] Alternatively, the host cell has been engineered such that anendogenous GnT III gene is activated, for example, by insertion of a DNAelement which increases gene expression into the host chromosome. In apreferred embodiment, the endogenous GnTIII has been activated byinsertion of a promoter, an enhancer, a transcription factor bindingsite, a transposon, or a retroviral element or combinations thereof intothe host cell chromosome. In another aspect, the host cell has beenselected to carry a mutation triggering expression of an endogenousGnTIII. Preferably, the host cell is the CHO cell mutant lec 10.

[0016] In a further preferred embodiment of the claimed invention, theat least one nucleic acid encoding a GnTIII is operably linked to aconstitutive promoter element.

[0017] In a further preferred embodiment, the host cell is a CHO cell, aBHK cell, a NSO cell, a SP2/0 cell, or a hybridoma cell, a YO myelomacell, a P3X63 mouse myeloma cell, a PER cell or a PER.C6 cell and saidpolypeptide is an anti-CD20 antibody. In another preferred embodiment,the host cell is a SP2/0 cell and the polypeptide is the monoclonalantibody chG250.

[0018] In another aspect, the claimed invention is directed to a hostcell that further comprises at least one transfected nucleic acidencoding an antibody molecule, an antibody fragment, or a fusion proteinthat includes a region equivalent to the Fc region of an immunoglobulin.In a preferred embodiment, the host cell comprises at least onetransfected nucleic acid encoding an anti-CD20 antibody, the chimericanti-human neuroblastoma monoclonal antibody chCE7, the chimericanti-human renal cell carcinoma monoclonal antibody chG250, the chimericanti-human colon, lung, and breast carcinoma monoclonal antibody ING-1,the humanized anti-human 17-1A antigen monoclonal antibody 3622W94, thehumanized anti-human colorectal tumor antibody A33, the anti-humanmelanoma antibody directed against GD3 ganglioside R24, or the chimericanti-human squamous-cell carcinoma monoclonal antibody SF-25, ananti-human EGFR antibody, an anti-human EGFRvIII antibody, an anti-humanPSMA antibody, and anti-human PSCA antibody, an anti-human CD22antibody, an anti-human CD30 antibody, an anti-human CD33 antibody, ananti-human CD38 antibody, an anti-human CD40 antibody, an anti-humanCD45 antibody, an anti-human CD52 antibody, an anti-human CD138antibody, an anti-human HLA-DR variant antibody, an anti-human EpCAMantibody, an anti-human CEA antibody, an anti-human MUC1 antibody, ananti-human MUC1 core protein antibody, an anti-human aberrantlyglycosylated MUCI antibody, an antibody against human fibronectinvariants containing the ED-B domain, and an anti-human HER2/neuantibody.

[0019] In another aspect, the claimed invention is directed to a methodfor producing a polypeptide in a host cell comprising culturing any ofthe above-described the host cells under conditions which permit theproduction of said polypeptide having increased Fc-mediated cellularcytotoxicity. In a preferred embodiment, the method further comprisesisolating said polypeptide having increased Fc-mediated cellularcytotoxicity.

[0020] In a further preferred embodiment, the host cell comprises atleast one nucleic acid encoding a fusion protein comprising a regionequivalent to a glycosylated Fc region of an immunoglobulin.

[0021] In a preferred embodiment, the proportion of bisectedoligosaccharides in the Fc region of said polypeptides is greater than50%, more preferably, greater than 70%. In another embodiment, theproportion of bisected hybrid oligosaccharides or galactosylated complexoligosaccharides or mixtures thereof in the Fc region is greater thanthe proportion of bisected complex oligosaccharides in the Fc region ofsaid polypeptide.

[0022] In a preferred aspect of the claimed method, the polypeptide isan anti-CD20 antibody and the anti-CD20 antibodies produced by said hostcell have a glycosylation profile, as analyzed by MALDI/TOF-MS, that issubstantially equivalent to that shown in FIG. 2E.

[0023] In another preferred aspect of the claimed method, thepolypeptide is the chG250 monoclonal antibody and the chG250 antibodiesproduced by said host cell have a glycosylaton profile, as analyzed byMALDI/TOF-MS, that is substantially equivalent to that shown in FIG. 7D.

[0024] In a further aspect, the claimed invention is directed to anantibody having increased antibody dependent cellular cytotoxicity(ADCC) produced by any of the methods described above. In preferredembodiments, the antibody is selected from the group consisting of ananti-CD20 antibody, chCE7, ch-G250, a humanized anti-HER2 monoclonalantibody, ING-1, 3622W94, SF-25, A33, and R24. Alternatively, thepolypeptide can be an antibody fragment that includes a regionequivalent to the Fc region of an immunoglobulin, having increasedFc-mediated cellular cytotoxicity produced by any of the methodsdescribed above.

[0025] In a further aspect, the claimed invention is directed to afusion protein that includes a region equivalent to the Fe region of animmunoglobulin and having increased Fc-mediated cellular cytotoxicityproduced by any of the methods described above.

[0026] In a further aspect, the claimed invention is directed to apharmaceutical composition comprising the antibody, antibody fragment,or fusion protein of the invention and a pharmaceutically acceptablecarrier.

[0027] In a further aspect, the claimed invention is directed to amethod for the treatment of cancer comprising administering atherapeutically effective amount of said pharmaceutical composition to apatient in need thereof.

[0028] In a further aspect, the invention is directed to an improvedmethod for treating an autoimmune disease produced in whole or in partby pathogenic autoantibodies based on B-cell depletion comprisingadministering a therapeutically effective amount of immunologicallyactive antibody to a human subject in need thereof, the improvementcomprising administering a therapeutically effective amount of anantibody having increased ADCC prepared as described above. In apreferred embodiment, the antibody is an anti-CD20 antibody. Examples ofautoimmune diseases or disorders include, but are not limited to,immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpurea and chronic idiopathic thrombocytopenicpurpurea, dermatomyositis, Sydenham's chorea, lupus nephritis, rheumaticfever, polyglandular syndromes, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, erythema multiform, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, primarybiliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronicactive hepatitis, polymyositis/dermnatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, polymyaglia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g. atopic dermatitis); systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); respiratory distresssyndrome (including adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoidarthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g. Type 1 diabetes mellitus or insulin dependent diabetesmellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious amenia (Addison's disease); diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder, multiple organ injury syndrome; hemolytic anemia (including,but not limited to cryoglobinemia or Coombs positive anemia);myastheniagravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter'sdisease; stiff-man syndrome; Behcet disease; giant cell arteritis;immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc. Inthis aspect of the invention, the antibodies of the invention are usedto deplete the blood of normal B-cells for an extended period.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1. Indirect immunofluorescence assay showing the reactivityof the antibody preparation C2B8-25t to CD20 positive SB cells. Negativecontrols, including the HSB CD20 negative cell line and cells treatedonly with the secondary FITC-conjugated anti-human Fc polyclonalantibody are not shown.

[0030]FIG. 2A-2E. MALDI/TOF-MS spectra of the oligosaccharides derivedfrom Mabthera™ (FIG. 2A), C2B8-nt (FIG. 2B), C2B8-2000t (FIG. 2C),C2B8-50t (FIG. 2D), and C2B8-25t (FIG. 2E) antibody samples.Oligosaccharides appear as [M+Na⁺] and [M+K⁺] ions. Oligosaccharideappearing in the first two spectra were derived from cell cultures thatdo not express GnTIII, whereas oligosaccharides in C, D, and E werederived from a single cell line expressing GnTIII at different levels(i.e. tetracycline concentrations). FIG. 3A and 3B. Illustration of atypical human IgG Fc-associated oligosaccharide structure (A) andpartial N-linked glycosylation pathway (B). FIG. 3A) The core of theoligosaccharide is composed of three mannose (M) and twoN-acetylglucosamine (Gn) monosaccharide residues attached to Asn₂₉₇.Galactose (G), fucose (F), and bisecting N-acetylglucosamine (Gn, boxed)can be present or absent. Terminal N-acetylneuraminic acid may be alsopresent but it is not included in the figure. (FIG. 3B) Partial N-linkedglycosylation pathway leading to the formation of the majoroligosaccharide classes (dotted frames). Bisecting N-acetylglucosamineis denoted as Gn^(b). Subscript numbers indicate how many monosaccharideresidues are present in each oligosaccharide. Each structure appearstogether with its sodium-associated [M+Na⁺] mass. The mass of thosestructures that contain fucose (f) are also included.

[0031]FIG. 4A and 4B. ADCC activities of Rituximab glycosylationvariants. The percentage of cytotoxicity was measured via lysis of ⁵¹Crlabeled CD20-positive SB cells by human lymphocytes (E:T ratio of 100:1)mediated by different mAb concentrations. (FIG. 4A) Activity of C2B8samples derived from a single cell line but produced at increasingGnTIII expression levels (i.e., decreasing tetracycline concentrations).The samples are C2B8-2000t, C2B8-50t, C2B8-25t, and C2B8-nt (control mAbderived from a clone that does not express GnTIII (FIG. 4B) ADCCactivity of C2B8-50t and C2B8-25t compared to Mabthera™.

[0032]FIG. 5. Western blot analysis of the seven GnTIII expressingclones and the wild type. 30 μg of each sample were loaded on a 8.75%SDS gel, transferred to a PVDF membrane and probed with the anti-c-mycmonoclonal antibody (9E10). WT refers to wt-chG250-SP2/0 cells.

[0033]FIG. 6. SDS polyacrylamide gel electrophoresis of resolvedpurified antibody samples.

[0034]FIG. 7A-7D. MALDI/TOF-MS spectra of neutral oligosaccharidemixtures from chG250 mAb samples produced by clones expressing differentGnTIII levels and wt-chG250-SP2/0 cells: WT (FIG. 7A), 2F1 (FIG. 7B),3D3 (FIG. 7C), 4E6 (FIG. 7D).

[0035]FIG. 8A-8D. MALDI/TOF-MS spectra of neutral oligosaccharidemixtures from chG250 mAb samples produced by clones expressing differentGnTIII levels: 4E8, (FIG. 8A); 5G2, (FIG. 8B); 4G3, (FIG. 8C); 5H12,(FIG. 8D).

[0036]FIG. 9. In vitro ADCC assay of antibody samples derived fromcontrol wt-chG250-SP2/-cells and GnTIII transected clones 3D3 and 5H12.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Terms are used herein as generally used in the art, unlessotherwise defined as follows:

[0038] As used herein, the term antibody is intended to include wholeantibody molecules, antibody fragments, or fusion proteins that includea region equivalent to the Fc region of an immunoglobulin.

[0039] As used herein, the term region equivalent to the Fc region of animmunoglobulin is intended to include naturally occurring allelicvariants of the Fc region of an immunoglobulin as well as variantshaving alterations which produce substitutions, additions, or deletionsbut which do not decrease substantially the ability of theimmunoglobulin to mediate antibody dependent cellular cytotoxicity. Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity.(See, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990).

[0040] As used herein, the term glycoprotein-modifying glycosyltransferase refers to β(1,4)-N-acetylglucosaminyltransferase III(GnTIII).

[0041] As used herein, the terms engineer, engineered, engineering andglycosylation engineering are considered to include any manipulation ofthe glycosylation pattern of a naturally occurring polypeptide orfragment thereof. Glycosylation engineering includes metabolicengineering of the glycosylation machinery of a cell, including geneticmanipulations of the oligosaccharide synthesis pathways to achievealtered glycosylation of glycoproteins expressed in cells. Furthermore,glycosylation engineering includes the effects of mutations and cellenvironment on glycosylation.

[0042] As used herein, the term host cell covers any kind of cellularsystem which can be engineered to generate modified glycoforms ofproteins, protein fragments, or peptides of interest, includingantibodies and antibody fragments. Typically, the host cells have beenmanipulated to express optimized levels of GnT III. Host cells includecultured cells, e.g., mammalian cultured cells, such as CHO cells, BHKcells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER. C6 cells or hybridoma cells, yeast cells, andinsect cells, to name only a few, but also cells comprised within atransgenic animal or cultured tissue.

[0043] As used herein, the term Fc-mediated cellular cytotoxicityincludes antibody-dependent cellular cytotoxicity and cellularcytotoxicity mediated by a soluble Fc-fusion protein containing a humanFc-region. It is an immune mechanism leading to the lysisof“antibody-targeted cells” by “human immune effector cells”, wherein:

[0044] The “human immune effector cells” are a population of leukocytesthat display Fc receptors on their surface through which they bind tothe Fc-region of antibodies or of Fc-fusion proteins and performeffector functions. Such a population may include, but is not limitedto, peripheral blood mononuclear cells (PBMC) and/or natural killer (NK)cells.

[0045] The “antibody-targeted cells” are cells bound by the antibodiesor Fc-fusion proteins. The antibodies or Fc fusion-proteins bind totarget cells via the protein part N-terminal to the Fc region.

[0046] As used herein, the term increased Fc-mediated cellularcytotoxicity is defined as either an increase in the number of“antibody-targeted cells” that are lysed in a given time, at a givenconcentration of antibody, or of Fc-fusion protein, in the mediumsurrounding the target cells, by the mechanism of Fc-mediated cellularcytotoxicity defined above, and/or a reduction in the concentration ofantibody, or of Fc-fusion protein, in the medium surrounding the targetcells, required to achieve the lysis of a given number of“antibody-targeted cells”, in a given time, by the mechanism of Fc-mediated cellular cytotoxicity. The increase in Fc-mediated cellularcytotoxicity is relative to the cellular cytotoxicity mediated by thesame antibody, or Fc-fusion protein, produced by the same type of hostcells, using the same standard production, purification, formulation andstorage methods, which are known to those skilled in the art, but thathas not been produced by host cells engineered to express theglycosyltransferase GnTIII by the methods described herein.

[0047] By antibody having increased antibody dependent cellularcytotoxicity (ADCC) is meant an antibody having increased ADCC asdetermined by any suitable method known to those of ordinary skill inthe art. One accepted in vitro ADCC assay is as follows:

[0048] 1) the assay uses target cells that are known to express thetarget antigen recognized by the antigen-binding region of the antibody;

[0049] 2) the assay uses human peripheral blood mononuclear cells(PBMCs), isolated from blood of a randomly chosen healthy donor, aseffector cells;

[0050] 3) the assay is carried out according to following protocol:

[0051] i) the PBMCs are isolated using standard density centriftigationprocedures and are suspended at 5×10⁶ cells/ml in RPMI cell culturemedium;

[0052] ii) the target cells are grown by standard tissue culturemethods, harvested from the exponential growth phase with a viabilityhigher than 90%, washed in RPMI cell culture medium, labelled with 100micro-Curies of ⁵¹Cr, washed twice with cell culture medium, andresuspended in cell culture medium at a density of 10⁵ cells/ml;

[0053] iii) 100 microliters of the final target cell suspension aboveare transferred to each well of a 96-well microtiter plate;

[0054] iv) the antibody is serially-diluted from 4000 ng/ml to 0.04ng/ml in cell culture medium and 50 microliters of the resultingantibody solutions are added to the target cells in the 96-wellmicrotiter plate, testing in triplicate various antibody concentrationscovering the whole concentration range above;

[0055] v) for the maximum release(MR) controls, 3 additional wells inthe plate containing the labelled target cells, receive 50 microlitersof a 2% (V/V) aqueous solution of non-ionic detergent (Nonidet, Sigma,St. Louis), instead of the antibody solution (point iv above);

[0056] vi) for the spontaneous release (SR) controls, 3 additional wellsin the plate containing the labelled target cells, receive 50microliters of RPMI cell culture medium instead of the antibody solution(point iv above);

[0057] vii) the 96-well microtiter plate is then centrifuged at 50×g for1 minute and incubated for 1 hour at 4° C.;

[0058] viii) 50 microliters of the PBMC suspension (point i above) areadded to each well to yield an effector: target cell ratio of 25:1 andthe plates are placed in an incubator under 5% CO₂ atmosphere at 37° C.for 4 hours;

[0059] ix) the cell-free supernatant from each well is harvested and theexperimentally released radioactivity (ER) is quantified using a gammacounter;

[0060] x) the percentage of specific lysis is calculated for eachantibody concentration according to the formula (ER-MR)/(MR-SR)×100,where ER is the average radioactivity quantified (see point ix above)for that antibody concentration, MR is the average radioactivityquantified (see point ix above) for the MR controls (see point v above),and SR is the average radioactivity quantified (see point ix above) forthe SR controls (see point vi above);

[0061] 4) “increased ADCC” is defined as either an increase in themaximum percentage of specific lysis observed within the antibodyconcentration range tested above, and/or a reduction in theconcentration of antibody required to achieve one half of the maximumpercentage of specific lysis observed within the antibody concentrationrange tested above. The increase in ADCC is relative to the ADCC,measured with the above assay, mediated by the same antibody, producedby the same type of host cells, using the same standard production,purification, formulation and storage methods, which are known to thoseskilled in the art, but that has not been produced by host cellsengineered to overexpress the glycosyltransferase GnTIII.

[0062] As used herein, the term anti-CD20 antibody is intended to meanan antibody which specifically recognizes a cell surfacenon-glycosylated phosphoprotein of 35,000 Daltons, typically designatedas the human B lymphocyte restricted differentiation antigen Bp35,commonly referred to as CD20.

[0063] Identification and Generation of Nucleic Acids Encoding A Proteinfor which Modification of the Glycosylation Pattern is Desired

[0064] The present invention provides methods for the generation and useof host cell systems for the production of glycoforms of antibodies orantibody fragments or fusion proteins which include antibody fragmentswith increased antibody-dependent cellular cytotoxicity. Identificationof target epitopes and generation of antibodies having potentialtherapeutic value, for which modification of the glycosylation patternis desired, and isolation of their respective coding nucleic acidsequence is within the scope of the invention.

[0065] Various procedures known in the art may be used for theproduction of antibodies to target epitopes of interest. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments and fragments produced by an Fab expressionlibrary. Such antibodies may be useful, e.g., as diagnostic ortherapeutic agents. As therapeutic agents, neutralizing antibodies,i.e., those which compete for binding with a ligand, substrate oradapter molecule, are of especially preferred interest.

[0066] For the production of antibodies, various host animals areimmunized by injection with the target protein of interest including,but not limited to, rabbits, mice, rats, etc. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,saponin, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum.

[0067] Monoclonal antibodies to the target of interest may be preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include, but arenot limited to, the hybridoma technique originally described by Kohlerand Milstein, Nature 256:495-97 (1975), the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72 (1983); Cote et al.,Proc. Natl. Acad. Sci. U.S.A. 80:2026-30 (1983 ) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy 77-96(Alan R. Liss, Inc., 1985)). In addition, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad.Sci. U.S.A. 81:6851-55 (1984); Neuberger et al., Nature 312:604-08(1984); Takeda et al., Nature 314:452-54 (1985) by splicing the genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce single chain antibodies having a desiredspecificity.

[0068] Antibody fragments which contain specific binding sites of thetarget protein of interest may be generated by known techniques. Forexample, such fragments include, but are not limited to, F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed (Huse et al., Science246:1275-81 (1989) to allow rapid and easy identification of monoclonalFab fragments with the desired specificity to the target protein ofinterest.

[0069] Once an antibody or antibody fragment has been identified forwhich modification in the glycosylation pattern are desired, the codingnucleic acid sequence is identified and isolated using techniques wellknown in the art.

[0070] a. Generation of Cell Lines for the Production of Proteins withAltered Glycosylation Pattern

[0071] The present invention provides host cell expression systems forthe generation of proteins having modified glycosylation patterns. Inparticular, the present invention provides host cell systems for thegeneration of glycoforms of proteins having an improved therapeuticvalue. Therefore, the invention provides host cell expression systemsselected or engineered to increase the expression of aglycoprotein-modifying glycosyltransferase, namelyβ(1,4)-N-acetylglucosaminyltransferase III (GnTIID). Specifically, suchhost cell expression systems may be engineered to comprise a recombinantnucleic acid molecule encoding GnTIII, operatively linked to aconstitutive or regulated promoter system. Alternatively, host cellexpression systems may be employed that naturally produce, are inducedto produce, and/or are selected to produce GnTIII.

[0072] In one specific embodiment, the present invention provides a hostcell that has been engineered to express at least one nucleic acidencoding GnTIII. In one aspect, the host cell is transformed ortransfected with a nucleic acid molecule comprising at least one geneencoding GnTIII. In an alternate aspect, the host cell has beenengineered and/or selected in such way that endogenous GnTIII isactivated. For example, the host cell may be selected to carry amutation triggering expression of endogenous GnTIII. In one specificembodiment, the host cell is a CHO lec 10 mutant. Alternatively, thehost cell may be engineered such that endogenous GnTIII is activated. Inagain another alternative, the host cell is engineered such thatendogenous GnTIII has been activated by insertion of a constitutivepromoter element, a transposon, or a retroviral element into the hostcell chromosome.

[0073] Generally, any type of cultured cell line can be used as abackground to engineer the host cell lines of the present invention. Ina preferred embodiment, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YOmyeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, yeast cells, or insect cells are used as the backgroundcell line to generate the engineered host cells of the invention.

[0074] The invention is contemplated to encompass any engineered hostcells expressing GnTIII as defined herein.

[0075] One or several nucleic acids encoding GnTIII may be expressedunder the control of a constitutive promoter or, alternately, aregulated expression system. Suitable regulated expression systemsinclude, but are not limited to, a tetracycline-regulated expressionsystem, an ecdysone-inducible expression system, a lac-switch expressionsystem, a glucocorticoid-inducible expression system, atemperature-inducible promoter system, and a metallothioneinmetal-inducible expression system. If several different nucleic acidsencoding GnTIII are comprised within the host cell system, some of themmay be expressed under the control of a constitutive promoter, whileothers are expressed under the control of a regulated promoter. Themaximal expression level is considered to be the highest possible levelof stable GnTIII expression that does not have a significant adverseeffect on cell growth rate, and will be determined using routineexperimentation Expression levels are determined by methods generallyknown in the art, including Western blot analysis using a GnTIIIspecific antibody, Northern blot analysis using a GnTIII specificnucleic acid probe, or measurement of enzymatic activity. Alternatively,a lectin may be employed which binds to biosynthetic products of theGnTIII, for example, E₄-PHA lectin. In a further alternative, thenucleic acid may be operatively linked to a reporter gene; theexpression levels of the GnTIII are determined by measuring a signalcorrelated with the expression level of the reporter gene. The reportergene may transcribed together with the nucleic acid(s) encoding saidGnTIII as a single mRNA molecule; their respective coding sequences maybe linked either by an internal ribosome entry site (IRES) or by acap-independent translation enhancer (CITE). The reporter gene may betranslated together with at least one nucleic acid encoding said GnTIIIsuch that a single polypeptide chain is formed. The nucleic acidencoding the GnTIII may be operatively linked to the reporter gene underthe control of a single promoter, such that the nucleic acid encodingthe GnTIII and the reporter gene are transcribed into an RNA moleculewhich is alternatively spliced into two separate messenger RNA (mRNA)molecules; one of the resulting mRNAs is translated into said reporterprotein, and the other is translated into said GnTIII.

[0076] If several different nucleic acids encoding GnTIII are expressed,they may be arranged in such way that they are transcribed as one or asseveral mRNA molecules. If they are transcribed as a single mRNAmolecule, their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). They may be transcribed from a single promoter into anRNA molecule which is alternatively spliced into several separatemessenger RNA (mRNA) molecules, which then are each translated intotheir respective encoded GnTIII.

[0077] In other embodiments, the present invention provides host cellexpression systems for the generation of therapeutic antibodies, havingan increased antibody-dependent cellular cytotoxicity, and cells whichdisplay the IgG Fc region on the surface to promote Fc-mediatedcytotoxicity. Generally, the host cell expression systems have beenengineered and/or selected to express nucleic acids encoding theantibody for which the production of altered glycoforms is desired,along with at least one nucleic acid encoding GnTIII. In one embodiment,the host cell system is transfected with at least one gene encodingGnTIII. Typically, the transfected cells are selected to identify andisolate clones that stably express the GnTIII. In another embodiment,the host cell has been selected for expression of endogenous GnTIII. Forexample, cells may be selected carrying mutations which triggerexpression of otherwise silent GnTIII. For example, CHO cells are knownto carry a silent GnT III gene that is active in certain mutants, e.g.,in the mutant Lec10. Furthermore, methods known in the art may be usedto activate silent GnTIII, including the insertion of a regulated orconstitutive promoter, the use of transposons, retroviral elements, etc.Also the use of gene knockout technologies or the use of ribozymemethods may be used to tailor the host cell's GnTIII expression level,and is therefore within the scope of the invention.

[0078] Any type of cultured cell line can be used as background toengineer the host cell lines of the present invention. In a preferredembodiment, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myelomacells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridomacells, yeast cell, or insect cells may be used. Typically, such celllines are engineered to further comprise at least one transfectednucleic acid encoding a whole antibody molecule, an antibody fragment,or a fusion protein that includes a region equivalent to the Fc regionof an immunoglobulin. In an alternative embodiment, a hybridoma cellline expressing a particular antibody of interest is used as backgroundcell line to generate the engineered host cells of the invention.

[0079] Typically, at least one nucleic acid in the host cell systemencodes GnT III.

[0080] One or several nucleic acids encoding GnTIII may be expressedunder the control of a constitutive promoter, or alternately, aregulated expression system Suitable regulated expression systemsinclude, but are not limited to, a tetracycline-regulated expressionsystem, an ecdysone-inducible expression system, a lac-switch expressionsystem, a glucocorticoid-inducible expression system, atemperature-inducible promoter system, and a metallothioneinmetal-inducible expression system. If several different nucleic acidsencoding GnTIII are comprised within the host cell system, some of themmay be expressed under the control of a constitutive promoter, whileothers are expressed under the control of a regulated promoter. Themaximal expression level is considered to be the highest possible levelof stable GnTIII expression that does not have a significant adverseeffect on cell growth rate, and will be determined using routineexperimentation. Expression levels are determined by methods generallyknown in the art, including Western blot analysis using a GnTIIIspecific antibody, Northern blot analysis using a GnTIII specificnucleic acid probe, or measurement of GnTIII enzymatic activity.Alternatively, a lectin may be employed which binds to biosyntheticproducts of GnTIII, for example, E₄-PHA lectin. In a furtheralternative, the nucleic acid may be operatively linked to a reportergene; the expression levels of the glycoprotein-modifying glycosyltransferase are determined by measuring a signal correlated with theexpression level of the reporter gene. The reporter gene may transcribedtogether with the nucleic acid(s) encoding said glycoprotein-modifyingglycosyl transferase as a single mRNA molecule; their respective codingsequences may be linked either by an internal ribosome entry site (IRES)or by a cap-independent translation enhancer (CITE). The reporter genemay be translated together with at least one nucleic acid encodingGnTIII such that a single polypeptide chain is formed. The nucleic acidencoding the GnTIII may be operatively linked to the reporter gene underthe control of a single promoter, such that the nucleic acid encodingthe GnTIII and the reporter gene are transcribed into an RNA moleculewhich is alternatively spliced into two separate messenger RNA (mRNA)molecules; one of the resulting mRNAs is translated into said reporterprotein, and the other is translated into said GnTIII.

[0081] If several different nucleic acids encoding a GnTIII areexpressed, they may be arranged in such way that they are transcribed asone or as several mRNA molecules. If they are transcribed as single mRNAmolecule, their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). They may be transcribed from a single promoter into anRNA molecule which is alternatively spliced into several separatemessenger RNA (mRNA) molecules, which then are each translated intotheir respective encoded GnTIII.

[0082] i. Expression Systems

[0083] Methods which are well known to those skilled in the art can beused to construct expression vectors containing the coding sequence ofthe protein of interest and the coding sequence of the GnTIII andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y (1989).

[0084] A variety of host-expression vector systems maybe utilized toexpress the coding sequence of the protein of interest and the codingsequence of the GnTIII. Preferably, mammalian cells are used as hostcell systems transfected with recombinant plasmid DNA or cosmid DNAexpression vectors containing the coding sequence of the protein ofinterest and the coding sequence of the GnTIII. Most preferably, CHOcells, ByIK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER. C6 cells or hybridoma cells, yeast cells,or insect cells are used as host cell system. In alternate embodiments,other eukaryotic host cell systems may be contemplated, including, yeastcells transformed with recombinant yeast expression vectors containingthe coding sequence of the protein of interest and the coding sequenceof the GnTIII; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the coding sequence ofthe protein of interest and the coding sequence of the GnTIII; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence of the protein of interest andthe coding sequence of the GnTIII; or animal cell systems infected withrecombinant virus expression vectors (e.g., adenovirus, vaccinia virus)including cell lines engineered to contain multiple copies of the DNAencoding the protein of interest and the coding sequence of the GnTIIIeither stably amplified (CHO/dhfr) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines).

[0085] For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

[0086] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guaninephosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectivelyAlso, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler et al., Natl.Acad. Sci. USA 77:3567 (1989); O'Hare et al., Proc. Natl. Acad. Sci. USA78:1527 (1981)); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,J Mol. Biol. 150:1 (1981)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)); the glutaminesynthase system; and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL- ornithine, DFMO (McConlogue, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.(1987)).

[0087] ii. Identification of Transfectants or Transformants that Expressthe Protein having a Modified Glycosylation Pattern

[0088] The host cells which contain the coding sequence and whichexpress the biologically active gene products may be identified by atleast four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b)the presence or absence of “marker” gene functions; (c) assessing thelevel of transcription as measured by the expression of the respectivemRNA transcripts in the host cell; and (d) detection of the gene productas measured by immunoassay or by its biological activity.

[0089] In the first approach, the presence of the coding sequence of theprotein of interest and the coding sequence of the GnTIII inserted inthe expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the respective coding sequences, respectively, or portionsor derivatives thereof

[0090] In the second approach, the recombinant expression vector/hostsystem can be identified and selected based upon the presence or absenceof certain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the coding sequence of the protein of interest and the codingsequence of the GnTIII are inserted within a marker gene sequence of thevector, recombinants containing the respective coding sequences can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the coding sequences under thecontrol of the same or different promoter used to control the expressionof the coding sequences. Expression of the marker in response toinduction or selection indicates expression of the coding sequence ofthe protein of interest and the coding sequence of the GnTIII.

[0091] In the third approach, transcriptional activity for the codingregion of the protein of interest and the coding sequence of the GnTIIIcan be assessed by hybridization assays. For example, RNA can beisolated and analyzed by Northern blot using a probe homologous to thecoding sequences of the protein of interest and the coding sequence ofthe GnTIII or particular portions thereof Alternatively, total nucleicacids of the host cell may be extracted and assayed for hybridization tosuch probes.

[0092] In the fourth approach, the expression of the protein products ofthe protein of interest and the coding sequence of the GnTIII can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active geneproducts.

[0093] b. Generation and Use of Proteins and Protein Fragments havingAltered Glycosylation Patterns

[0094] I. Generation and Use of Antibodies having IncreasedAntibody-Dependent Cellular Cytotoxicity

[0095] In preferred embodiments, the present invention providesglycoforms of antibodies and antibody fragments having increasedantibody-dependent cellular cytotoxicity.

[0096] Clinical trials of unconjugated monoclonal antibodies (mAbs) forthe treatment of some types of cancer have recently yielded encouragingresults. Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); Deo etal., Immunology Today 18:127 (1997). A chimeric, unconjugated IgG1 hasbeen approved for low-grade or follicular B-cell non-Hodgkin's lymphomaDillman, Cancer Biother. & Radiopharm. 12:223-25 (1997), while anotherunconjugated mAb, a humanized IgG1 targeting solid breast tumors, hasalso been showing promising results in phase III clinical trials. Deo etal., Immunology Today 18:127 (1997). The antigens of these two mAbs arehighly expressed in their respective tumor cells, and the antibodiesmediate potent tumor destruction by effector cells in vitro and in vivo.In contrast, many other unconjugated mAbs with fine tumor specificitiescannot trigger effector functions of sufficient potency to be clinicallyuseful. Frost et al., Cancer 80:317-33 (1997), Surfus et al., J.Immunother. 19:184-91 (1996). For some of these weaker mAbs, adjunctcytokine therapy is currently being tested. Addition of cytokines canstimulate antibody-dependent cellular cytotoxicity (ADCC) by increasingthe activity and number of circulating lymphocytes. Frost et al., Cancer80:317-33 (1997); Surfus et al., J. Immunother. 19:184-91 (1996). ADCC,a lytic attack on antibody-targeted cells, is triggered upon binding ofleukocyte receptors to the constant region (Fc) of antibodies. Deo etal., Immunology Today 18:127 (1997)

[0097] A different, but complementary, approach to increase ADCCactivity of unconjugated IgG1s is to engineer the Fc region of theantibody to increase its affinity for the lymphocyte receptors (FcγRs).Protein engineering studies have shown that FcγRs interact with thelower hinge region of the IgG CH2 domain. Lund et al., J. Immunol.157:4963-69 (1996). However, FcγR binding also requires the presence ofoligosaccharides covalently attached at the conserved Asn 297 in the CH2region. Lund et al., J. Immunol. 157:4963-69 (1996); Wright andMorrison, Trends Biotech. 15:26-31 (1997), suggesting that eitheroligosaccharide and polypeptide both directly contribute to theinteraction site or that the oligosaccharide is required to maintain anactive CH2 polypeptide conformation. Modification of the oligosaccharidestructure can therefore be explored as a means to increase the affinityof the interaction.

[0098] An IgG molecule carries two N-linked oligosaccharides in its Fcregion, one on each heavy chain. As any glycoprotein, an antibody isproduced as a population of glycoforms which share the same polypeptidebackbone but have different oligosaccharides attached to theglycosylation sites. The oligosaccharides normally found in the Fcregion of serum IgG are of complex bi-antennary type (Wormald et al.,Biochemistry 36:130-38 (1997), with low level of terminal sialic acidand bisecting N-acetylglucosamine (GIcNAc), and a variable degree ofterminal galactosylation and core fucosylation. Some studies suggestthat the minimal carbohydrate structure required for FcγR binding lieswithin the oligosaccharide core. Lund et al., J. Immunol. 157:4963-69(1996) The removal of terminal galactoses results in approximately atwo-fold reduction in ADCC activity, indicating a role for theseresidues in FcγR receptor binding. Lund et al., J. Immunol. 157:4963-69(1996)

[0099] The mouse- or hamster-derived cell lines used in industry andacademia for production of unconjugated therapeutic mAbs normally attachthe required oligosaccharide determinants to Fc sites. IgGs expressed inthese cell lines lack, however, the bisecting GIcNAc found in lowamounts in serum IgGs. Lifely et al., Glycobiology 318:813-22 (1995). Incontrast, it was recently observed that a rat myeloma-produced,humanized IgG1 (CAMPATH-1H) carried a bisecting GlcNAc in some of itsglycoforms. Lifely et al., Glycobiology 318:813-22 (1995). The ratcell-derived antibody reached a similar in vitro ADCC activity asCAMPATH-1H antibodies produced in standard cell lines, but atsignificantly lower antibody concentrations.

[0100] The CAMPATH antigen is normally present at high levels onlymphoma cells, and this chimeric mAb has high ADCC activity in theabsence of a bisecting GlcNAc. Lifely et al., Glycobiology 318:813-22(1995). In the N-linked glycosylation pathway, a bisecting GlcNAc isadded by the enzyme β(1,4)-N-acetylglucosaminyltransferase III (GnTIII). Schachter, Biochem. Cell Biol. 64:163-81 (1986).

[0101] The present inventors used a single antibody-producing CHO cellline, that was previously engineered to express, in anexternally-regulated fashion, different levels of a cloned GnT III gene.This approach established for the first time a rigorous correlationbetween expression of GnTIII and the ADCC activity of the modifiedantibody.

[0102] The present inventors previously showed that C2B8 antibodymodified according to the disclosed method had an about sixteen-foldhigher ADCC activity than the standard, unmodified C2B8 antibodyproduced under identical cell culture and purification conditions.Briefly, a C2B8 antibody sample expressed in CHO-tTA-C2B8 cells that donot have GnTIII expression showed a cytotoxic activity of about 31% (at1 μg/ml antibody concentration), measured as in vitro lysis of SB cells(CD20+) by human lymphocytes. In contrast, C2B8 antibody derived from aCHO cell culture expressing GnT III at a basal, largely repressed levelshowed at 1 μg/ml antibody concentration a 33% increase in ADCC activityagainst the control at the same antibody concentration. Moreover,increasing the expression of GnT III produced a large increase of almost80% in the maximal ADCC activity (at 1 μg/ml antibody concentration)compared to the control at the same antibody concentration. (SeeInternational Publication No. WO 99/54342, the entire contents of whichare hereby incorporated by reference.)

[0103] Further antibodies of the invention having increasedantibody-dependent cellular cytotoxicity include, but are not limitedto, anti-human neuroblastoma monoclonal antibody (chCE7) produced by themethods of the invention, a chimeric anti-human renal cell carcinomamonoclonal antibody (ch-G250) produced by the methods of the invention,a humanized anti-HER2 monoclonal antibody (e.g., Trastuzumab(HERCEPTIN)) produced by the methods of the invention, a chimericanti-human colon, lung, and breast carcinoma monoclonal antibody (ING-1)produced by the methods of the invention, a humanized anti-human 17-1Aantigen monoclonal antibody (3622W94) produced by the methods of theinvention, a humanized anti-human colorectal tumor antibody (A33)produced by the methods of the invention, an anti-human melanomaantibody (R24) directed against GD3 ganglioside produced by the methodsof the invention, and a chimeric anti-human squamous-cell carcinomamonoclonal antibody (SF-25) produced by the methods of the invention, ananti-human small cell lung carcinoma monoclonal antibody (BEC2, ImCloneSystems, Merck KgaA) produced by the methods of the invention, ananti-human non-Hodgkin's lymphoma monoclonal antibody (Bexxar(tositumomab, Coulter Pharmaceuticals), Oncolym (Techniclone, AlphaTherapeutic)) produced by the methods of the invention, an anti-humansquamous cell head and neck carcinoma monoclonal antibody (C225, ImCloneSystems) prepared by the methods of the invention, an anti-human rectaland colon carcinoma monoclonal antibody (Panorex (edrecolomab),Centocor, Glaxo Wellcome) prepared by the methods of the invention, ananti-human ovarian carcinoma monoclonal antibody (Theragyn, Antisoma)produced by the methods of the invention, an anti-human acutemyelogenous leukemia carcinoma monoclonal antibody (SmartM195, ProteinDesign Labs, Kanebo) produced by the methods of the invention, ananti-human malignant glioma monoclonal antibody (Cotara, Techniclone,Cambridge Antibody Technology) produced by the methods of the invention,an anti-human B cell non-Hodgkins lymphoma monoclonal antibody(IDEC-Y2B8, IDEC Pharmaceuticals) produced by the methods of theinvention, an anti-human solid tumors monoclonal antibody (CEA-Cide,Immunomedics) produced by the methods of the invention, an anti-humancolorectal carcinoma monoclonal antibody (Iodine 131-MN-14,Immunomedics) produced by the methods of the invention, an anti-humanovary, kidney, breast, and prostate carcinoma monoclonal antibody(MDX-210, Medarex, Novartis) produced by the methods of the invention,an anti-human colorectal and pancreas carcinoma monoclonal antibody(TTMA, Pharmacie & Upjohn) produced by the methods of the invention, ananti-human TAG-72 expressing carcinoma monoclonal antibody (MDX-220,Medarex) produced by the methods of the invention, an anti-humanEGFr-expressing carcinoma monoclonal antibody (MDX-447) produced by themethods of the invention, Anti-VEGF monoclonal antibody (Genentech)produced by the methods of the invention, an anti-human breast, lung,prostate and pancreas carcinoma and malignant melanoma monoclonalantibody (BrevaRex, AltaRex) produced by the methods of the invention,and an anti-human acute myelogenous leukemia monoclonal antibody(Monoclonal Antibody Conjugate, Immunex) produced by the methods of theinvention. In addition, the invention is directed to antibody fragmentand fusion proteins comprising a region that is equivalent to the Fcregion of immunoglobulins.

[0104] ii. Generation and Use of Fusion Proteins Comprising a RegionEquivalent to an Fc Region of an Immunoglobulin that Promote Fc-MediatedCytotoxicity

[0105] As discussed above, the present invention relates to a method forincreasing the ADCC activity of therapeutic antibodies. This is achievedby engineering the glycosylation pattern of the Fc region of suchantibodies, in particular by maximizing the proportion of antibodymolecules carrying bisected complex oligosaccharides and bisected hybridoligosaccharides N-linked to the conserved glycosylation sites in theirFc regions. This strategy can be applied to increase Fc-mediatedcellular cytotoxicity against undesirable cells mediated by any moleculecarrying a region that is an equivalent to the Fc region of animmunoglobulin, not only by therapeutic antibodies, since the changesintroduced by the engineering of glycosylation affect only the Fc regionand therefore its interactions with the Fc receptors on the surface ofeffector cells involved in the ADCC mechanism. Fc-containing moleculesto which the presently disclosed methods can be applied include, but arenot limited to, (a) soluble fusion proteins made of a targeting proteindomain fused to the N-terminus of an Fc-region (Chamov and Ashkenazi,Trends Biotech. 14: 52 (1996) and (b) plasma membrane-anchored fusionproteins made of a type II transmembrane domain that localizes to theplasma membrane fused to the N-terminus of an Fc region (Stabila, P. F.,Nature Biotech. 16: 1357 (1998)).

[0106] In the case of soluble fusion proteins (a) the targeting domaindirects binding of the fusion protein to undesirable cells such ascancer cells, i.e., in an analogous fashion to therapeutic antibodies.The application of presently disclosed method to enhance the Fc-mediatedcellular cytotoxic activity mediated by these molecules would thereforebe identical to the method applied to therapeutic antibodies.

[0107] In the case of membrane-anchored fusion proteins (b) theundesirable cells in the body have to express the gene encoding thefusion protein. This can be achieved either by gene therapy approaches,i.e., by transfecting the cells in vivo with a plasmid or viral vectorthat directs expression of the fusion protein-encoding gene toundesirable cells, or by implantation in the body of cells geneticallyengineered to express the fusion protein on their surface. The latercells would normally be implanted in the body inside a polymer capsule(encapsulated cell therapy) where they cannot be destroyed by anFc-mediated cellular cytotoxicity mechanism. However should the capsuledevice fail and the escaping cells become undesirable, then they can beeliminated by Fc-mediated cellular cytotoxicity. Stabila et al., NatureBiotech. 16: 1357 (1998). In this case, the presently disclosed methodwould be applied either by incorporating into the gene therapy vector anadditional gene expression cassette directing adequate or maximalexpression levels of GnT III or by engineering the cells to be implantedto express adequate or maximal levels of GnT III. In both cases, the aimof the disclosed method is to increase or maximize the proportion ofsurface-displayed Fc regions carrying bisected complex oligosaccharidesand/or bisected hybrid oligosaccharides.

[0108] The examples below explain the invention in more detail. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

EXAMPLE 1

[0109] New Versions of the Chimeric Anti-CD20 Antibody IDEC-C2B8 HavingEnhanced Antibody-Dependent Cellular Cytotoxicity Obtained byGlycosylation Engineering of an IDEC-CEB8 Producing Cell Line

[0110] Synthesis of VHand VL coding regions of IDEC-C2B8 andconstruction of mammalian expression vectors. cDNAs encoding the VH andVL regions of IDEC-C2B8 antibody were assembled from a set ofoverlapping single-stranded oligonucleotides in a one-step process usingPCR (Kobayashi, N., et al., Biotechniques 23:500-503 (1997)). Theoriginal sequence data coding for IDEC-C2B8 VL and VH were obtained froma published international patent application (International PublicationNumber: WO 94/11026). Assembled VL and VH cDNA fragments were subclonedinto pBluescriptIIKS(+), sequenced and directly joined by ligation tothe human constant light (Igκ) and heavy (IgG1) chain cDNAs,respectively, using unique restriction sites introduced at the variableand constant region junctions without altering the original amino acidresidue sequence (Umana, P., et al., Nat Biotechnol. 17:176-180 (1999);Reff, M. E., et al., Blood 83:435-445 (1994)). Each full-length cDNA wasseparately subcloned into pcDNA3.1(+) (Invitrogen, Leek, TheNetherlands) yielding mammalian expression vectors for chimeric C2B8light (pC2B8L) and heavy (pC2B8H) chains.

[0111] Production of IDEC-C2B8 in CHO cells expressing different levelsof GnTIII. Establishment of two CHO cell lines, CHO-tet-GnTIIIexpressing different levels of GnTIII depending on the tetracyclineconcentration in the culture medium; and CHO-tTA, the parental cell linethat does not express GnTIII has been described previously (Umana, P.,et al., Nat Biotechnol. 17:176-180 (1999); Umana, P., et al., BiotechnolBioeng. 65:542-549 (1999)). Each cell line was cotranfected with vectorspC2B8L, pC2B8H, and pZeoSV2(+) (for Zeocin resistance; Invitrogen, Leek,The Netherlands) using a calcium phosphate method. Zeocin resistantclones were transferred to a 96-well plate and assayed for IDEC-C2B8production using an ELISA assay specific for the human constant region(4). Three IDEC-C2B8 samples were obtained from parallel cultures of aselected clone (CHO-tet-GnTIII-C2B8), differing only in the tetracyclineconcentration added to the medium (25, 50 and 2000 ng/mL respectively).Culture supernatants were harvested in the late exponential phase. Anadditional antibody sample was obtained from a CHO-tTA-derived clone,CHO-tTA-C2B8, cultured under identical conditions but without addingtetracycline to the medium. Antibody samples were purified from culturemedium by protein A affinity chromatography and buffer exchanged to PBSon a cation exchange column as previously described (Umana, P., et al.,Nat Biotechnol. 17:176-180 (1999)). Antibody concentration was measuredusing a fluorescence-based kit from Molecular Probes (Leiden, TheNetherlands) with Rituximab used as standard.

[0112] Indirect immunofluorescence. CD20-positive cells (SB cells; ATCCdeposit no. ATCC CCL120) and CD20-negative cells (HSB cells; ATCCdeposit no. ATCC CCL120.1) were each incubated for 1h with 2.5 μg/ml ofCHO-tet-GnTIII-derived IDEC-C2B8 antibody in Hank's balanced saltsolution (GibcoBRL, Basel, Switzerland) and 2% bovine serum albuminfraction V (Roche Diagnostics, Rotkreuz, Switzerland) (HBSSB). As anegative control HBSSB was used instead of C2B8 antibody. AFITC-conjugated, anti-human Fc polyclonal antibody was used as asecondary antibody (SIGMA, St. Louis) for all samples. Cells wereexamined using a Leica fluorescence microscope (Wetzlar, Germany).

[0113] Oligosaccharide profiling by MALDI/TOF-MS. Neutral, N-linkedoligosaccharides were derived from C2B8 antibody samples, MabThera™(European counterpart of Rituximab; kind gift from R. Stahel,Universit{dot over (a)}tspital, Switzerland), C2B8-25t, C2B8-50t,C2B8-2000t, and C2B8-nt, (100 μg each) as previously described (Umana,P., et al., Nat Biotechnol. 17:176-180 (1999)). Briefly, the antibodysamples were first treated with Arthrobacter ureafaciens sialidase(Oxford Glycosciences, Abingson, UK) to remove any sialic acidmonosaccharide residues. Neutral N-linked oligosaccharides were thenreleased from the desialylated antibody samples usingpeptide-N-glycosidase F (Oxford Glycosciences), purified usingmicro-columns, and analyzed by MALDI/TOF-MS in an Elite Voyager 400spectrometer (Perseptive Biosystems, Farmingham, Mass.).

[0114] ADCC Activity Assay. Peripheral blood mononuclear cells (PBMC)were separated from heparinated fresh human blood (in all experimentsobtained from the same healthy donor) by centrifugation over aFicoll-Paque (Pharmacia Biotech, Dübendorf, Switzerland) gradient. PBMC(effector) were depleted of monocytes by plastic adherence.CD20-positive SB (target) cells, were labeled for 90 min with 100 μCi⁵¹Cr (Amersham, Dübendorf, Switzerland) at 37° C., washed twice in RPMI(GibcoBRL, Basel, Switzerland) and resuspended at a concentration of 10⁵cells/ml. Fifty microliters of C2B8 mAb diluted in RPMI medium was addedto 100 μl SB cells (10,000 cells/well) in a 96-well round bottommicrotiter plate (Greiner, Langenthal, Switzerland), centrifuged at 50×gfor 1 min, and incubated for 1 h at 4° C. Subsequently, 50 μl ofeffector cell (suspended at 2×10⁷ cells/ml in RPMI medium) were added toeach 96-well yielding a final E:T ratio of 100. Plates were incubatedfor 4 h at 37° C. and 5% CO₂, supernatant was harvested with a Skatronharvesting system (Skatron Instruments, Sterling, Va.) and counted (ER,experimental release) in a Cobra 05005 γ counter (Canberra Packard,Meriden, Conn.). Maximum (MR) and spontaneous (SR) releases wereobtained by adding, instead of C2B8 mAb, 100 μl of 1% Nonidet (Sigma,St. Louis) or 100 μl of RPMI medium, respectively, to 100 μl labeledtarget cells. All data points were performed in triplicate. Specificlysis (%) was calculated with the following formula:(ER-SR)/(Mk-SR)×100.

[0115] Results and Discussion

[0116] Production of IDEC-C2B8 and verification of specific antigenbinding. CHO-tet-GnTIII cells, with stable, tetracycline-regulatedexpression of GnTIII and stable, constitutive expression of IDEC-C2B8,were established and scaled-up for production of a set of antibodysamples. During scale-up, parallel cultures from the same clone weregrown under three different tetracycline concentrations, 25, 50 and 2000ng/ml. These levels of tetracycline had previously been shown to resultin different levels of GnTIII and bisected oligosaccharides (Umana, P.,et al., Nat Biotechnol. 17:176-180(1999); Umana, P., et al., BiotechnolBioeng. 65:542-549 (1999)). A C2B8-producing, control cell line thatdoes not express GnTIII was also established and cultured under the sameconditions as for the three parallel cultures of CHO-tet-GnTIII. AfterProtein A-affinity chromatography, mAb purity was estimated to be higherthan 95% by SDS-PAGE and Coomassie-blue staining. The samples were namedaccording to the tetracycline concentration added to the culture mediumfor their production: C2B8-25t, C2B 8-50t, C2B8-2000t and C2B8-nt (i.e.,no tetracycline for the non-bisected control). Sample C2B8-25t showedspecific antigen binding by indirect immunofluorescence usingCD20-positive and CD20-negative cells (FIG. 1), indicating that thesynthesized VL and VH gene fragments were functionally correct.

[0117] Oligosaccharide profiling with MALDI/TOF-MS. The glycosylationprofile of each antibody sample was analyzed by MALDI/TOF-MS of thereleased, neutral oligosaccharide mix. In this technique,oligosaccharides of different mass appear as separate peaks in thespectrum and their proportions are quantitatively reflected by therelative peak heights (Harvey, D. J., Rapid Common Mass Spectrom.7:614-619 (1993); Harvey, D. J., et al., Glycoconj J. 15:333-338(1998)).Oligosaccharide structures were assigned to different peaks based ontheir expected molecular masses, previous structural data foroligosaccharides derived from IgGI mAbs produced in the same host, andinformation on the N-linked oligosaccharide biosynthetic pathway.

[0118] A clear correlation was found between GnTIII expression levels(i.e., tetracycline concentration) and the amount of bisectedoligosaccharides derived from the different antibody samples. Asexpected, MabThera™ and C2B8-nt, which are derived from hosts that donot express GnTIII, did not carry bisected oligosaccharides (FIGS. 2Aand 2B). In contrast, bisected structures amounted up to approximately35% of the oligosaccharides pool in sample C2B8-2000t, i.e, at a basallevel of GnTIII expression. In this case, the main bisectedoligosaccharide peaks were of complex type, unequivocally assigned topeaks at m/z 1689 and m/z 1851 (FIG. 2C). The next higher GnTIIIexpression level, sample C2B8-50t, led to an increase in these peaks(including their associated potassium aducts at m/z 1705 and 1861) ofaround 20%. This increase was accompanied by a concomitant reduction oftheir non-bisected counterparts at m/z 1486 and 1648, respectively (FIG.2D). At the highest GnTIII expression level, sample C2B8-25t, the mainsubstrate for GnTIII, m/z 1486, decreased to almost base-line level,while complex bisected structures (m/z 1689 and 1851) decreased in favorof increases in peaks at mn/z 1664, 1810 and 1826 (FIG. 2E). These peakscan be assigned either to bisected hybrid compounds, to galactosylatedcomplex oligosaccharides, or to a mixture of both. Their relativeincrease, however, is consistent with the accumulation of bisectedhybrid compounds, as GnTIII over expression can divert the biosyntheticflux at early stages of the pathway (see FIG. 3A and 3B). The amount ofbisected oligosaccharide structures (complex and hybrid type) reachedapproximately 80% for this sample.

[0119] ADCC activity of IDEC-C2B8 glycosylated variants. Different C2B8mAb glycosylation variants were compared for ADCC activity, measured asin vitro lysis of CD20-positive SB cells. An additional mAb sample,C2B8-nt, derived from the parental cell line lacking GnTIII, was alsostudied. Sample C2B8-2000t produced at the basal GnTIII expression leveland carrying low levels of bisected oligosaccharides was slightly moreactive than C2B8-nt (FIG. 4A). At the next higher level of GnTIII-expression, sample C2B8-50t carried approximately equal levels ofbisected and non-bisected oligosaccharides, but did not mediatesignificantly higher target-cell lysis. However, at the lowesttetracycline concentration, sample C2B8-25t, which contained up to 80%of bisected oligosaccharide structures, was significantly more activethan the rest of the samples in the whole antibody concentration range.It reached the maximal level of ADCC activity of sample C2B8-nt at a10-fold lower antibody concentration (FIG. 4A). Sample C2B8-25t alsoshowed a significant increase in the maximal ADCC activity with respectto the control (50% vs. 30% lysis).

[0120] Samples C2B8-50t and C2B8-25t, bearing the highest proportions ofbisected oligosaccharides, were further compared in ADCC activity toMabthera™, the version of Rituxan™ currently marketed in Europe (FIG.4B). Sample C2B8-50t showed a slight increase in activity whereas sampleC2B8-25t clearly out performed Mabthera™ at all antibody concentrations.Approximately a five to ten-fold lower concentration of C2B8-25t wasrequired to reach the maximal ADCC activity of Mabthera™, and themaximal activity of C2B8-25t was about 25% higher than that ofMabthera™.

[0121] These results show that, in general, the in vitro ADCC activityof the C2B8 antibody correlates with the proportion of moleculescarrying bisected oligosaccharides in the Fc region. We had previouslyreported that in the case of chCE7, an antibody with a low baselinelevel of ADCC activity, significant increases of activity could beobtained by increasing the fraction of bisected oligosaccharides abovethe levels found in naturally-occurring antibodies (Umana, P., et al.,Nat Biotechnol. 17:176-180 (1999)). The same is true for the C2B8 mAb,which already has high ADCC activity in the absence of bisectedoligosaccharides. In the case of chCE7, however, very large increases ofADCC activity were observed at a level of GnTIII expression wherebisected oligosaccharides were predominantly of complex type (Umana, P.,et al., Nat Biotechnol. 17:176-180 (1999)). For the potent C2B8 mAb,such a large boost in activity was only observed at the highest levelsof GnTIII expression studied, where bisected oligosaccharides hadshifted mainly to the hybrid type (FIG. 2). For both mAbs, the sampleswith the highest activities had considerably higher levels of bisectedthan non-bisected oligosaccharides. Together, these observationsindicate that probably both complex and hybrid bisected oligosaccharidesare important for ADCC activity.

[0122] In both complex and hybrid oligosaccharides, a bisecting GlcNAcleads to a large change in oligosaccharide conformations (Balaji, P. V.,et al., Int. J. Biol. Macromol. 18:101-114 (1996)). The change occurs ina part of the oligosaccharide that interacts extensively with thepolypeptide in the CH2 domain (Jefferis, R., et al., Immunol Rev.163:59-76 (1998)). Since the polypetide is relatively flexible at thislocation (Jefferis, R., et al., Immunol Rev. 163:59-76 (1998)), it ispossible that the bisecting N-acetylglucosamine is mediating itsbiological effects through a conformational change in the Fc region. Thepotentially altered conformations would already exist in nature, as allserum IgGs carry bisected oligosaccharides. The main difference betweenthe engineered and natural antibodies would be the proportion ofmolecules displaying the more active conformations.

[0123] Various approaches for increasing the activity of unconjugatedmAbs are currently under clinical evaluation, includingradio-immunotherapy, antibody-dependent enzyme/prodrug therapy,immunotoxins and adjuvant therapy with cytokines (Hjelm Skog, A., etal., Cancer Immunol Immunother. 48:463-470 (1999); Blakey, D. C., etal., Cell Biophys. 25:175-183 (1994); Wiseman, G. A., et al., ClinCancer Res. 5:3281s-3296s (1999); Hank, J. A., et al., Cancer Res.50:5234-5239 (1990)). These technologies can give large increases inactivity, but they can also lead to significantly higher side effects,elevated production costs and complex logistics from production toadministration to patients when compared to unconjugated mAbs. Thetechnology presented here offers an alternative way to obtain increasesin potency while maintaining a simple production process, and should beapplicable to many unconjugated mAbs.

EXAMPLE 2

[0124] New Versions of the Anti-Renal Cell Carcinoma Antibody chG250Having Enhanced Antibody-Dependent Cellular Cytotoxicity Obtained byGlycosylation Engineering of a chG250 Producing Cell Line

[0125] 1. Cell Culture

[0126] SP2/0 mouse myeloma cells producing chG250 chimeric mAb(wt-chG250-SP2/0 cells) were grown in standard cell culture mediumsupplemented with 1:100 (v/v) penicillin/streptomycin/antimycoticsolution (SIGMA, Buchs, Switzerland). Cells were cultured at 37° C. in a5% CO₂ humidified atmosphere in Tissue Culture Flasks. Medium waschanged each 3-4 days. Cells were frozen in culture medium containing10% DMSO.

[0127] 2. Generation of SP2/0 Cells with pGnTIII-Puro Expression

[0128] wt-chG250-SP2/0 myeloma cells were transfected by electroporationwith a vector for constitutive expression of GnTIII operatively linkedvia an IRES to a puromycin resistance gene. 24 hours beforeelectroporation culture medium was changed and cells were seeded at5×10⁵ cells/ml. Seven million cells were centrifuged for 4 min at 1300rpm at 4° C. Cells were washed with 3 mL new medium and centrifugedagain. Cells were resuspended in a volume of 0.3-0.5 ml of reaction mix,containing 1.25% (v/v) DMSO and 20-30 μg DNA in culture medium. Theelectroporation mix was then transferred to a 0.4 cm cuvette and pulsedat low voltage (250-300 V) and high capacitance (960 μF) using GenePulser from Bio Rad. After electroporation cells were quicklytransferred to 6 mL 1.25% (v/v) DMSO culture medium in a T25 cultureflask and incubated at 37° C. Stable integrants were selected byapplying 2 μg/mL puromycin to the medium two days after electroporation.After 2-3 weeks a stable, puromycin-resitant mixed population wasobtained. Single-cell derived clones were obtained via FACS and weresubsequently expanded and maintained under puromycin selection.

[0129] 3. Western Blot

[0130] Puromycin-resistant clones were screened for GnTIII expression byWestern blotting. The Western blots clearly showed that clones 5H12, 4E6and 4E8 were expressing the highest levels of GnTIII. 5G2 also showed aGnTIII band of middle intensity, whereas 2F1, 3D3 and 4G3 had the lowestband intensities, therefore expressing lower amounts of GnTIII (FIG. 5).

[0131] 4. Production and Purification of chG250 Monoclonal Antibody fromSeven GnTIII-Expressing Clones Including Wild Type

[0132] Clones 2F1, 3D3, 4E6, 4E8, 4G3, 5G2, 5H12 and the wild type(wt-chG250-SP2/0 cells) were seeded at 3×10⁵ cells/mL in a total volumeof 130 ml culture medium, and cultivated in single Triple-flasks. Cellsused for seeding were all in full exponential growth phase, thereforecells were considered to be at the same growth state when the productionbatches started. Cells were cultivated for 4 days. Supernatantscontaining the antibody were collected in the late exponential growthphase to ensure reproducibility . The chG250 monoclonal antibody waspurified in two chromatographic steps. Culture supernatants containingthe chG250 monoclonal antibody derived from each batch were firstpurified using a HiTrap Protein A affinity chromatography. Protein A ishighly specific for the human IgG F_(c) region. Pooled samples from theprotein A eluate were buffer exchanged to PBS by cation-exchangechromatography on a Resource S 1 ml column (Amersham Pharmacia Biotech).Final purity was judged to be higher than 95% from SDS-staining andCoomassie blue staining (FIG. 6). The concentration of each sample wasdetermined with a standard calibration curve using wild type antibodywith known concentration.

[0133] 5. Oligosaccharide Profiling of mAb Preparations Derived from theSeven Clones Expressing Different GnTIII Levels

[0134] Oligosaccharide profiles were obtained by matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI/TOF-MS),which accurately provides the molecular masses of the differentoligosaccharide structures. This technique allows a quantitativeanalysis of proportions between different oligosacchaiide structureswithin a mixture. Neutral oligosaccharides appeared predominantly as[M+Na⁺] ions, however sometimes they were accompanied by smaller [M+K⁺]ions, leading to an increase in mass of m/z of 16. The percentage of thestructure appearing as potassium ion adducts depends on the content ofthe matrix and may thus vary between samples. A mixture of neutralN-linked oligosaccharides derived from each antibody preparation wasanalyzed using a 2,5-dehydrobenzoic acid (2,5-DHB) as matrix. Some ofthe peaks in the spectra were unequivocally assigned to specificoligosaccharide structures, because of known monosaccharide compositionand unique mass. However, sometimes multiple structures could beassigned to a particular mass. MALDI enables the determination of themass and cannot distinguish between isomers. Knowledge of thebiosynthetic pathway and previous structural data enable, in most cases,the assignment of an oligosaccharide structure to a peak in thespectrum.

[0135] Oligosaccharides derived from the mAb sample produced inwt-chG250-SP2/0 cell line, that does not express GnTIII, containednonbisected biantennary complex (m/z 1486) and mono- ordi-galactosylated nonbisected biantennary complex structures (FIG. 7A),both α(1,6)-fucosylated in the core region (peaks m/z 1648 and 1810respectively).

[0136] Expression of GCTIII generated bisected Fc-associatedoligosaccharide structures of two types: complex or hybrid. Complexbisected oligosaccharides were unequivocally assigned to peaks at m/z1543, 1689, 1705, 1851 and 1867 ([M+K⁺] adduct). As expected, theincrease in bisected oligosaccharides was accompanied by a concomitantreduction of peaks m/z 1486 and 1648, that correspond to nonbisectedcomplex oligosaccharides. For all samples derived from the GnTIIIexpressing clones, the main substrate of GnTIII (m/z 1486) decreaseddramatically. As expected, the percentage of the nonbisected complexoligosaccharide type, assigned to peak at m/z 1648, had the lowestvalues for the clones expressing the highest GnTIII levels (clones 4E6,4E8, 5G2 and 5H12). These two peaks decreased in favor of theaccumulation of bisected complex and bisected hybrid typeoligosaccharides (FIGS. 7A-7D and 8A-8D). The percentage of bisectedcomplex oligosaccharides was higher for the samples derived from theclones expressing lower amounts of GnTIII. This is consistent with thefact that a higher GnTIII expression level probably shifts thebiosynthetic flux to bisected hybrid structures, thereby decreasing therelative proportions of complex and complex bisected compound. Forbisected hybrid structures, two possible structures could sometimes beassigned to a single peak. Therefore, some assumptions were made inorder to approximate the percentage of these structures over the totaloligosaccharide pool. Peaks m/z 1664, 1680, 1810 and 1826 can beassigned to either bisected hybrid type, to galactosylated complexoligosaccharides, or a mixture of them. Due to the fact that thewt-antibody preparation had a relatively low percentage of peak 1664, itwas assumed that this peak, appearing in significant amounts in theantibody samples derived from the different clones, correspondedentirely to bisected hybrid structures (FIGS. 7A-7D and 8A-8D). Howeverto assign a specific structure to peaks m/z 1810 and 1826 furthercharacterization has to be performed. In summary, by over expression ofGnTIII, bisected oligosaccharides structures were generated and theirrelative proportions correlated with GnTIII expression levels.

[0137] 6. Measurement of Antibody Mediated Cytotoxic Activity byCalcein-AM Retention

[0138] The Calcein-AM retention method of measuring cytotoxicitymeasures the dye fluorescence remaining in the cells after incubationwith the antibody. Four million G250 antigen-positive cells (target)were labelled with 10 μM Calcein-AM (Molecular Probes, Eugene, Oreg.) in1.8 mL RPMI-1640 cell culture medium (GIBCO BRL, Basel, Switzerland)supplemented with 10% fetal calf serum for 30 min at 37° C. in a 5% CO₂humidified atmosphere. The cells were washed twice in culture medium andresuspended in 12 mL AIMV serum free medium (GIBCO BRL, Basel,Switzerland). Labelled cells were then transferred to U-bottom 96-wells(30,000 cells/well) and incubated in triplicate with differentconcentrations of antibody for 1 hour at 4° C. Peripheral bloodmononuclear cells (PBMC) were separated from heparinated fresh humanblood (in all experiments obtained from the same healthy donor) bycentrifugation over a Ficoll-Paque (Pharmacia Biotech, Dübendorf,Switzerland) gradient PBMCs were added in triplicate wells in a 50 μLvolume, yielding an effector to target ratio (E:T ratio) of 25:1 and afinal volume of 200 μL. The 96-well plate was then incubated for 4 hoursat 37° C. in a 5% CO₂ atmosphere. Thereafter the 96-well plate wascentrifuged at 700×g for 5 min and the supernatants were discarded. Thecell pellets were washed twice with Hank's balanced salt solution (HBSS)and lysed in 200 μL 0.05M sodium borate, pH 9, 0.1% Triton X-100.Retention of the fluorescent dye in the target cells was measured with aFLUO star microplate reader (BMG Lab Technologies, Offenburg, Germany).The specific lysis was calculated relative to a total lysis control,resulting from exposure of the target cells to saponin (200 μg/mL inAIMV; SIGMA, Buchs, Switzerland) instead of exposure to antibody.Specific lysis (%) was calculated with the following formula:${\% \quad {Cytotoxicity}} = \frac{F_{med} - F_{e\quad {xp}}}{F_{med} - F_{\det}}$

[0139] where F_(med) represents the fluorescence of target cells treatedwith medium alone and considers unspecific lysis by PMBCs, F_(exp)represents the fluorescence of cells treated with antibody and F_(det)represents the fluorescence of cells treated with saponin instead ofantibody.

[0140] To determine the effect of modified glycosylation variants ofchG250 on the in vitro ADCC activity, G250 antigen-positive target cellswere cultured with PBMCs with and without chG250 antibody samples atdifferent concentrations. The cytotoxicity of unmodified chG250 antibodyderived from the wild type cell line was compared with two antibodypreparations derived from two cell lines (3D3, 5H12) expressingintermediate and high GnTIII levels, respectively (see FIG. 5).

[0141] Unmodified chG250 antibody did not mediate significant ADCCactivity over the entire concentration range used in the assay (theactivity was not significantly different from background). AugmentedADCC activity (close to 20%, see FIG. 9) at 2 μg/mL was observed withthe antibody sample derived from clone 3D3, which expressed intermediateGnTIII levels. The cytotoxic activity of this antibody samples did notgrow at higher antibody concentrations. As expected the antibodypreparation derived from clone 5H12 showed a striking increase oversamples 3D3 and unmodified antibody in its ability to mediate ADCCagainst target cells. The maximal ADCC activity of this antibodypreparation was around 50% andwas remarkable in mediating significantADCC activity at 125-fold less concentrated when comparing with theunmodified control sample.

EXAMPLE 3

[0142] Treatment of Immune-Mediated Thrombocytopenia in a Patient withChronic Graft-Versus-Host Disease

[0143] Autoimmune thrombocytopenia in chronic graft-versus-host diseaserepresents an instance of B-cell dysregulation leading to clinicaldisease. To treat immune-mediated thrombocytopenia in a subject withchronic graft-versus-host disease, an anti-CD20 chimeric monoclonalantibody prepared by the methods of the present invention and havingincreased ADCC is administered to the subject as described inRatanatharathom, V. et al., Ann. Intern. Med. 133(4):275-79 (2000) (theentire contents of which is hereby incorporated by reference).Specifically, a weekly infusion of the antibody, 375 mg/² isadministered to the subject for 4 weeks. The antibody therapy produces amarked depletion of B cells in the peripheral blood and decreased levelsof platelet-associated antibody.

EXAMPLE 4

[0144] Treatment of Severe, Immune-Mediated, Pure Red Cell Aplasia andHemolytic Anemia

[0145] Immune-mediated, acquired pure red cell aplasia (PRCA) is a raredisorder frequently associated with other autoimmune phenomena. To treatimmune-mediated, acquired pure red cell aplasia in a subject, ananti-CD20 chimeric monoclonal antibody prepared by the methods of thepresent invention and having increased ADCC is administered to thesubject as described in Zecca, M. et al., Blood 12:3995-97 (1997) (theentire contents of which are hereby incorporated by reference).Specifically, a subject with PRCA and autoimmune hemolytic anemia isgiven two doses of antibody, 375 mg/m², per week. After antibodytherapy, substitutive treatment with intravenous immunoglobulin isinitiated. This treatment produces a marked depletion of B cells and asignificant rise in reticulocyte count accompanied by increasedhemoglobin levels.

[0146] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0147] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

What is claimed is:
 1. A host cell engineered to produce a polypeptidehaving increased Fc-mediated cellular cytotoxicity by expression of atleast one nucleic acid encoding β(1,4)-N-acetylglucosaminyltransferaseIII (GnT III), wherein said polypeptide produced by said host cell isselected from the group consisting of a whole antibody molecule, anantibody fragment, and a fusion protein which includes a regionequivalent to the Fc region of an immunoglobulin, and wherein said GnTIII is expressed in an amount sufficient to increase the proportion ofsaid polypeptides carrying bisected hybrid oligosaccharides orgalactosylated complex oligosaccharides or mixtures thereof in the Fcregion relative to polypeptides carrying bisected complexoligosaccharides in the Fc region.
 2. The host cell of claim 1, whereinsaid polypeptide is IgG or a fragment thereof.
 3. The host cell of claim1, wherein said polypeptide is IgG1 or a fragment thereof.
 4. The hostcell of claim 1, wherein said polypeptide is a fusion protein thatincludes a region equivalent to the Fc region of a human IgG.
 5. Thehost cell of claim 1, wherein a nucleic acid molecule comprising atleast one gene encoding GnTIII has been introduced into said host cell.6. The host cell of claim 1, wherein said host cell has been engineeredsuch that an endogenous GnT III gene is activated.
 7. The host cell ofclaim 6, wherein said endogenous GnTIII has been activated by insertionof a DNA element which increases gene expression into the hostchromosome.
 8. The host cell of claim 6, wherein said host cell has beenselected to carry a mutation triggering expression of an endogenousGnTIII.
 9. The host cell of claim 8, wherein said host cell is the CHOcell mutant lec
 10. 10. The host cell of claim 1, wherein said host cellis a CHO cell, a BHK cell, a NS0 cell, a SP2/0 cell, a YO myeloma cell,a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell or a hybridomacell.
 11. The host cell of claim 10, wherein said polypeptide is ananti-CD20 antibody.
 12. The host cell of claim 11, wherein saidanti-CD20 antibody is IDEC-C2B8.
 13. The host cell of claim 10, whereinsaid host cell is a SP2/0 cell.
 14. The host cell of claim 13, whereinsaid antibody is the chimeric anti-human renal cell carcinoma monoclonalantibody chG250.
 15. The host cell of claim 5, wherein said at least onegene encoding GnTIII has been introduced into said host cell chromosome.16. The host cell of claim 6, wherein said endogenous GnTIII has beenactivated by insertion of a promoter element, a transposon, or aretroviral element into the host cell chromosome.
 17. The host cell ofclaim 1, further comprising at least one transfected nucleic acidencoding an antibody molecule, an antibody fragment, or a fusion proteinthat includes a region equivalent to the Fc region of an immunoglobulin.18. The host cell of claim 1, wherein said at least one nucleic acidencoding a GnTIII is operably linked to a constitutive promoter element.19. The host cell of claim 17, wherein said host cell comprises at leastone transfected nucleic acid encoding an anti-CD20 antibody, thechimeric anti-human neuroblastoma monoclonal antibody chCE7, thechimeric anti-human renal cell carcinoma monoclonal antibody chG250, thechimeric anti-human colon, lung, and breast carcinoma monoclonalantibody ING-1, the humanized anti-human 17-1A antigen monoclonalantibody 3622W94, the humanized anti-human colorectal tumor antibodyA33, the anti-human melanoma antibody directed against GD3 gangliosideR24, the chimeric anti-human squamous-cell carcinoma monoclonal antibodySF-25, an anti-human EGFR antibody, an anti-human EGFRvIII antibody, ananti-human PSMA antibody, an anti-human PSCA antibody, an anti-humanCD22 antibody, an anti-human CD30 antibody, an anti-human CD33 antibody,an anti-human CD38 antibody, an anti-human CD40 antibody, an anti-humanCD45 antibody, an anti-human CD52 antibody, an anti-human CD138antibody, an anti-human BLA-DR variant antibody, an anti-human EpCAMantibody, an anti-human CEA antibody, an anti-human MUC1 antibody, ananti-human MUC1 core protein antibody, an anti-human aberrantlyglycosylated MUC1 antibody, an antibody against human fibronectinvariants containing the ED-B domain, or an anti-human HER2/neu antibody.20. A method for producing a polypeptide in a host cell comprisingculturing the host cell of any one of claims 1-19 under conditions whichpermit the production of said polypeptide having increased Fc-mediatedcellular cytotoxicity.
 21. The method of claim 20, further comprisingisolating said polypeptide having increased Fc-mediated cellularcytotoxicity.
 22. The method of claim 20, wherein said host cellcomprises at least one nucleic acid encoding a fusion protein comprisinga region equivalent to a Fc region of an immunoglobulin.
 23. The methodof claim 20, wherein greater than 50% of the oligosaccharides in the Fcregion of said polypeptides are bisected.
 24. The method of claim 20,wherein greater than 70% of the oligosaccharides in the Fc region ofsaid polypeptides are bisected.
 25. The method of claim 20, wherein theproportion of bisected hybrid oligosaccharides or galactosylated complexoligosaccharides or mixtures thereof in the Fc region is greater thanthe proportion of bisected complex oligosaccharides in the Fc region ofsaid polypeptides.
 26. The method of claim 20, wherein said polypeptideis the anti-CD20 antibody IDEC-C2B8 and the IDEC-C2B8 antibodiesproduced by said host cell have a glycosylaton profile, as analyzed byMALDI/TOF-MS, that is substantially equivalent to that shown in FIG. 2E.27. The method of claim 20, wherein said polypeptide is the chG250monoclonal antibody and the chG250 antibodies produced by said host cellhave a glycosylaton profile, as analyzed by MALDI/TOF-MS, that issubstantially equivalent to that shown in FIG. 7D.
 28. An antibodyhaving increased antibody dependent cellular cytotoxicity (ADCC)produced by the method of claim
 21. 29. The antibody of claim 28,wherein said antibody is selected from the group consisting ofIDEC-C2B8, chCE7, ch-G250, a humanized anti-HER2 monoclonal antibody,ING-1, 3622W94, SF-25, A33, and R24.
 30. An antibody fragment thatincludes a region equivalent to the Fc region of an immunoglobulin,having increased Fc-mediated cellular cytotoxicity produced by themethod of claim
 21. 31. A fusion protein that includes a regionequivalent to the Fc region of an immunoglobulin and having increasedFc-mediated cellular cytotoxicity produced by the method of claim 21.32. A pharmaceutical composition comprising the antibody of claim 28 anda pharmaceutically acceptable carrier.
 33. A pharmaceutical compositioncomprising the antibody fragment of claim 30 and a pharmaceuticallyacceptable carrier.
 34. A pharmaceutical composition comprising thefusion protein of claim 31 and a pharmaceutically acceptable carrier.35. A method for the treatment of cancer comprising administering atherapeutically effective amount of the pharmaceutical composition ofany one of claims 32-34 to a patient in need thereof.
 36. An improvedmethod for disease treatment based on B-cell depletion comprisingadministering a therapeutically effective amount of antibody to a humansubject in need thereof, the improvement comprising administering atherapeutically effective amount of an antibody produced by the methodof claim
 28. 37. The improved method of claim 36, wherein said antibodyis an anti-CD20 monoclonal antibody.
 38. The improved method of claim37, wherein said anti-CD20 antibody is IDEC-C2B8.